Novel 1,3,5-Triazinyl Aminobenzenesulfonamides Incorporating Aminoalcohol, Aminochalcone and Aminostilbene Structural Motifs as Potent Anti-VRE Agents, and Carbonic Anhydrases I, II, VII, IX, and XII Inhibitors

A series of 1,3,5-triazinyl aminobenzenesulfonamides substituted by aminoalcohol, aminostilbene, and aminochalcone structural motifs was synthesized as potential human carbonic anhydrase (hCA) inhibitors. The compounds were evaluated on their inhibition of tumor-associated hCA IX and hCA XII, hCA VII isoenzyme present in the brain, and physiologically important hCA I and hCA II. While the test compounds had only a negligible effect on physiologically important isoenzymes, many of the studied compounds significantly affected the hCA IX isoenzyme. Several compounds showed activity against hCA XII; (E)-4-{2-[(4-[(2,3-dihydroxypropyl)amino]-6-[(4-styrylphenyl)amino]-1,3,5-triazin-2-yl)amino]ethyl}benzenesulfonamide (31) and (E)-4-{2-[(4-[(4-hydroxyphenyl)amino]-6-[(4-styrylphenyl)amino]-1,3,5-triazin-2-yl)amino]ethyl}benzenesulfonamide (32) were the most effective inhibitors with KIs = 4.4 and 5.9 nM, respectively. In addition, the compounds were tested against vancomycin-resistant Enterococcus faecalis (VRE) isolates. (E)-4-[2-({4-[(4-cinnamoylphenyl)amino]-6-[(4-hydroxyphenyl)amino]-1,3,5-triazin-2-yl}amino)ethyl]benzenesulfonamide (21) (MIC = 26.33 µM) and derivative 32 (MIC range 13.80–55.20 µM) demonstrated the highest activity against all tested strains. The most active compounds were evaluated for their cytotoxicity against the Human Colorectal Tumor Cell Line (HCT116 p53 +/+). Only 4,4’-[(6-chloro-1,3,5-triazin-2,4-diyl)bis(iminomethylene)]dibenzenesulfonamide (7) and compound 32 demonstrated an IC50 of ca. 6.5 μM; otherwise, the other selected derivatives did not show toxicity at concentrations up to 50 µM. The molecular modeling and docking of active compounds into various hCA isoenzymes, including bacterial carbonic anhydrase, specifically α-CA present in VRE, was performed to try to outline a possible mechanism of selective anti-VRE activity.

Target compounds  were synthesized according to the methodology published in [38] by a step-by-step nucleophile substitution of chlorine atoms of 4,6-dichloro-1,3,5triazin-2-yl aminobenzenesulfhonamide. The appropriate starting triazinyl aminobenzenesulfonamide reacted with a nucleophile in the presence of anhydrous potassium carbonate in a molar ratio of 1:1:1. Reactions were catalyzed by Cu(I)-supported on a weakly acidic resin. The substitution of the second or third chlorine atom was controlled by the temperature mode (Scheme 2). Scheme 1. General scheme of synthesis of target chalcone precursors. Stilbene precursors (E-H) were synthesized by a slightly modified Wittig-Horner reaction [36,37].
Target compounds  were synthesized according to the methodology published in [38] by a step-by-step nucleophile substitution of chlorine atoms of 4,6-dichloro-1,3,5triazin-2-yl aminobenzenesulfhonamide. The appropriate starting triazinyl aminobenzenesulfonamide reacted with a nucleophile in the presence of anhydrous potassium carbonate in a molar ratio of 1:1:1. Reactions were catalyzed by Cu(I)-supported on a weakly acidic resin. The substitution of the second or third chlorine atom was controlled by the temperature mode (Scheme 2).

CA Inhibition
The presence of aminobenzenesulfonamide structural moiety in the structure of target compounds 1-44 can cause inhibition activity in these compounds against human carbonic anhydrases. The inhibition of physiologically relevant hCA I and hCA II can lead to various undesirable side effects. On the other hand, the inhibition of tumor-associated isoenzymes hCA IX and hCA XII could be a great benefit in tumor treatment. Inhibition of isoenzyme hCA VII, which is presented mostly in the brain, can be a new approach for the treatment of epilepsy [41,42] or chronic neuropathic pain [42,43]. Compounds 1-44 were evaluated as potential inhibitors of hCA I, hCA II, hCA VII, hCA IX, and hCA XII. The inhibition activities against individual hCAs were determined by methodology based on stoppedflow assay [44]. The obtained results are shown in Table 2. For completeness, Table 2 also includes the previously published inhibition activities of compounds 2, 3, 5-12 and 18-19. Values of K I s were compared with clinically used sulfonamides as standards-AAZ

CA Inhibition
The presence of aminobenzenesulfonamide structural moiety in the structure of target compounds 1-44 can cause inhibition activity in these compounds against human carbonic anhydrases. The inhibition of physiologically relevant hCA I and hCA II can lead to various undesirable side effects. On the other hand, the inhibition of tumor-associated -Scheme 2. General synthetic scheme for the synthesis of target compounds 1-44. Table 2. Inhibition data for synthesized s-triazine derivatives 1-44 (for the structure see Scheme 2) and standard sulfonamide CA inhibitors against hCA I, hCA II, hCA VII, hCA IX, and hCA XII; their selectivity ratios for inhibition of isozyme hCA VII over hCA II, hCA IX over hCA II, and hCA XII over hCA II. For the evaluation, a methodology based on stopped-flow assay was employed [45]; three different assays for each compound; see Section 4.3.  [46]; c [47]; d [48]; e [39]. Based on data presented in Table 2, some general conclusions about relationships between the structure and activity of tested compounds can be made:

•
All tested compounds are weak inhibitors of cytosolic isozyme hCA I with K I s in a range from 8.5 to >10,000 nM. In general, compounds substituted with stilbenes are better inhibitors of hCA I than compounds containing chalcone substituents. Three stilbene derivatives (31,33, and 42) with the highest activity against hCA I with K I s in the range of 36.9-48.0 nM have at the terminal benzene core (R 2 ) hydrogen (4-H) or hydroxyl functional group (4-OH) in the position para. If the substituents present on the triazine core are very bulky (for example, in a homologous series of compounds 4, 17, and 37), the compounds' inhibitory activity increases with the increasing number of CH 2 groups between the triazine core and the benzenesulfonamide structural moiety. If the methylene or ethylene group between the triazine and benzene core is not present, the compound does not fit into the narrowing cavity of the active site due to the steric inherence. • All compounds presented in this article are very weak inhibitors of physiologically relevant isoenzyme hCA II. In comparison with previously reported compounds 2, 3, 5-12 and 18-19, chalcone and stilbene derivatives exhibit much lower inhibition activity against hCA II. This is probably caused by the bulkiness of chalcone and stilbene structural moieties. , it can be assumed that biological activity and selectivity of chalcone derivatives are negatively affected by the increasing length of the alkyl chain between the 1,3,5-triazine core and the benzenesulfonamide moiety.

•
The inhibitory activities of compounds 2, 3, 5-12 and 18-19 against the isoenzyme hCA IX were published and discussed extensively in [39]. For this reason, only the values of K I s in the inhibition of hCA IX for chalcone and stilbene derivatives were discussed in this part. Although the structures of chalcone derivatives with potential inhibition activity against isoenzyme hCA IX were selected based on docking (please see the Supplementary Materials), none of the tested compounds showed significant biological activity or selectivity against this isoenzyme. Contrarily, four of tested stilbene derivatives (17,  implies that, with an increasing number of CH 2 groups in the aminobenzene sulfonamide structural moiety, the biological activity decreases. Thus, it seems that the bulkiness of the molecule is not the only essential factor that affects the biological activity of the tested substances. However, it does significantly affect their selectivity. These observations suggest that, not only does the interaction of the sulfonamide functional group with the active site influence activity and selectivity, but also that the interactions between the rest of the molecule and amino acid residues of the cavity or amino acid residues close to the cavity must have a major effect on the activity and selectivity. In accordance with this statement, it is remarkable that the compounds with excellent selectivity and, at the same time,

VRE Inhibition
All the investigated compounds were tested on their anti-staphylococcal activity against three clinical isolates of methicillin-resistant Staphylococcus aureus (MRSA) [49] and S. aureus ATCC 29213 as the reference and quality control strain. Surprisingly, these compounds did not show any anti-staphylococcal activity (>256 µg/mL), therefore the data are not reported. On the other hand, some compounds demonstrated a selective ability to inhibit the growth of enterococci. All the compounds were tested against Enterococcus faecalis ATCC 29212 as the reference strain and three isolates from American crows of vanA-carrying vancomycin-resistant E. faecalis (VRE) [50], and the activities, expressed as minimum inhibitory concentrations (MICs), are listed in Table 3.
Based on the presented results, it can be stated that derivatives 21 and 32 are the most effective compounds against all tested strains. Other compounds, such as 26, 9, and, optionally, 25, 24, 7, and 29, can also be considered active against at least two of the VRE isolates. It is evident that the activity is associated with substitution at the R 1 position with the 1-(4-hydroxyphenyl)amino fragment. It appears to be preferred that the compounds be substituted with a bulky lipophilic substituent represented by either a chalcone or stilbene fragment, the subsequent substitution of which rather decreases potency. In this case, it is also advantageous if the linker between the triazine and sulfonamide parts of the molecule is three-membered, which ensures higher conformational flexibility of both parts of the molecule. On the other hand, activity was also observed for derivative 9, where the bulky and lipophilic aromatic substituent is replaced by a hydrophilic 2,3-hydroxypropylamine chain. It should be noted that this modification of the basic scaffold increases the solubility. On the other hand, similarly substituted potent compounds are limited in amount, therefore it is not possible to decide whether this is a preferred structural modification of anti-VRE active compounds derived from 1,3,5-triazinyl aminobenzenesulfonamides.
structural moiety, the biological activity decreases. Thus, it seems that the bulkiness of the molecule is not the only essential factor that affects the biological activity of the tested substances. However, it does significantly affect their selectivity. These observations suggest that, not only does the interaction of the sulfonamide functional group with the active site influence activity and selectivity, but also that the interactions between the rest of the molecule and amino acid residues of the cavity or amino acid residues close to the cavity must have a major effect on the activity and selectivity. In accordance with this statement, it is remarkable that the compounds with excellent selectivity and, at the same time, inhibitory activities comparable with all standards, contain 4'-H-aminostilbene structural moiety 30 (KI = 7.   Based on the observed results, one may speculate about the specific effectivity against Enterococcus sp. It can be hypothesized that anti-VRE activity is caused by the inhibition of bacterial carbonic anhydrase, specifically α-CA, present in VRE. Unfortunately, the pure CA isoenzyme from VRE has not been isolated yet. Therefore, the mechanism of action cannot be experimentally confirmed. However, the selective antibacterial activity could indicate a special mechanism of action associated with the effect on species-specific CA, as discussed below. Table 3. Data of antimicrobial activity (MICs) against E. faecalis (EF) and three vancomycin-resistant strains of VRE in comparison with standard ampicillin (AMP) and vancomycin (VAN). The inhibition activity against enterococci was evaluated by the microtitration broth method, according to CLSI, with some modifications; the experiment was repeated at least three times; see Section 4.4.    The compounds with promising inhibitory activity against tumor-associated isoenzymes hCA IX and hCA XII or anti-VRE activity (7, 9, 20, 21, 24, 25, 26, 29, and 32) were investigated to observe whether they influenced the metabolic activity using the HCT116 p53 +/+ colorectal tumor cell line after 48 h treatment. Their influence was evaluated as an IC 50 value (concentration caused a decrease in metabolic activity to 50%). Doxorubicin was used as the positive control. The results are shown in Table 4. Treatment with 9, 20, 21, 24, 25, 26, and 29 in the highest concentration (50 µM) did not significantly decrease the metabolic activity of the investigated tumor cell line. Only compounds 7 and 32 influenced metabolic activity, and their IC 50 values were determined. Their ability to decrease metabolic activity is lower in comparison with doxorubicin. To conclude these results, compounds 9, 20, 21, 24, 25, 26, and 29 could be discussed as promising antibacterial agents, and compounds 7 and 32 could be further tested for their possible use as anticancer drugs. Table 4. Cytotoxicity data against Human Colorectal Tumour Cell Line (HCT116 p53 +/+ ). The cytotoxicity evaluation was performed only for compounds with promising inhibitory activity against hCA IX, hCA XII, or anti-VRE activity. For the evaluation, MTT assay was employed; each individual compound was tested in triplicate and repeated three times; see Section 4.5.

Molecular Docking into hCA IX
A small virtual combinatorial library of 76 compounds was used for virtual screening of hCA IX, and the computed score of the ligands (Supplementary Materials) served as the selection of compounds for synthesis and biological evaluation. The molecules for synthesis were selected from the virtual combinatorial library according to occurrence of substituents on the s-triazine core among the best-scoring ligands. For validation, the acetazolamide that co-crystallized with the hCA IX was re-docked in the protein structure with RMSD = 1.57 Å (Supplementary Materials Figure S3), showing good results of the used docking method.
Poses of those ligands with the best inhibition activity against hCA IX (4, 17, 30, 37, 38, 40, 41, 43) were optimized in the active site of a hCA IX crystal structure, and the interaction energies were computed. The values of the binding free energies of top poses obtained from molecular docking (Table 5) are in the range from −55.08 to −90.82 kcal/mol. These values predict strong binding to the hCA IX. The correlation with MIC is poor because of the very narrow range of both computed binding energies and concentrations (taking the expected error of both methods into account). All poses of the molecules showed an interaction between the deprotonated nitrogen of the sulfonamide group and Zn 2+ (Supplementary Materials Figure S1). Distance between nitrogen and Zn 2+ was in a range between 2.15 and 2.17 Å for the complexes with the lowest energy. An oxygen atom of the sulfonamide group created a longer coordination bond with the zinc dication (distance in the range 2.5-2.8 Å), forming a bidentate arrangement. The hydrogen atom of the sulfonamide group served as the H-bond with Thr-199 residue side-chain oxygen. At the same time, the second oxygen of the sulfonamide group served as an acceptor of the H-bond with Thr-199.
As shown in Figure 2, benzenesulfonamide substituents are oriented to the bottom of the cavity, where they can make coordination bonds with the zinc dication, and all the chalcone/stilbene substituents occupy the same space along the hydrophobic side of the cavity while the third substituent on triazine ring is oriented towards the polar side of the cavity. The arrangement of ligand 4 is slightly different. The reason is a short linker between the benzenesulfonamide and s-triazine rings. Ligands 15 and 25 have the same substituents but a longer linker between the benzenesulfonamide and s-triazine rings, therefore they accommodated the same volume as other ligands with a longer linker between the rings ( Figure 3). While all ligands with longer a linker interact with Trp-5 as H-bond acceptors from indole >NH group, some of them are even H-bond donors to the imidazole of the known proton shuttle residue His-64 by means of s-triazine bound >NH group bearing polar substituent or to the Ser-3 carbonyl oxygen (this interaction is weaker because of longer distance) by means of the s-triazine bound >NH group bearing a chalcone/stilbene moiety ( Figure 4). The short linker prevents such interaction in this set of ligands.    The s-triazine core bears three substituents responsible for different interactions of the ligand:

•
The first substituent is always the benzenesulfonamide moiety with linkers of three different lengths. The coordination of the sulfonamide group to the metal center usually makes up 60% of the interaction energy of the ligands with the carbonic anhydrase [51].

•
The second substituent is either stilbene or chalcone, making hydrophobic interaction, which is the weakest interaction, however is significant because of the big surface covered. Most of these substituents bear phenolic hydroxyl at the end, which usually creates H-bonds with bulk water, but in some cases does so with polar groups of amino acid residues at the edge of the cavity (ligand 37 with the hydroxyl of Ser-20; ligand 4 with the carbonyl of Val-19).  The s-triazine core bears three substituents responsible for different interactions of the ligand:

•
The first substituent is always the benzenesulfonamide moiety with linkers of three different lengths. The coordination of the sulfonamide group to the metal center usually makes up 60% of the interaction energy of the ligands with the carbonic anhydrase [51].

•
The second substituent is either stilbene or chalcone, making hydrophobic interaction, which is the weakest interaction, however is significant because of the big surface covered. Most of these substituents bear phenolic hydroxyl at the end, which usually creates H-bonds with bulk water, but in some cases does so with polar groups of amino acid residues at the edge of the cavity (ligand 37 with the hydroxyl of Ser-20; ligand 4 with the carbonyl of Val-19).

Retrieval of Enterococcal CA Protein Sequence
There were no resolved 3D structures available for the α-CA protein of E. faecalis in PDB yet. In the literature, it was reported that, for homology modeling, the sequence identity between target and template proteins should be 25% or more [52]. The crystal structure of CA from Thermovibrio ammonificans (4C3T) was selected with a 31% similarity sequence for the target sequence and 6% for gaps. The sequence alignment is depicted in Figure 9.

3D Homology Modeling
The stereochemical quality of the 3D model was validated by the Ramachandran plot. Figure 10 showed that around 97.8% of the residues were present in the allowed regions (88.4% in the favored region and 9.4% residues in the allowed regions) and that only 2.2% of residues were present in the outlier region, indicating that the quality of the model was good. The plot did not change significantly after MD (Molecular dynamic) simulation, and MD showed the stability of the structure (Figure 16, see Section 4.6.4 in Materials and Methods). backbone carbonyl of Asn-62 ( Figure 8). The aromatic ring of this phenol substituent also allows T-stacking with His-64.   Figure S2). The remaining hydrogen of the sulfonamide group serves as a donor for the H-bond with the backbone carbonyl oxygen of Leu-179 in all cases except ligand 21. While other ligands have the distance between hydrogen and oxygen atoms 2.4 Å, the top pose of ligand 21 shows a distance of 3.1 Å, and the pose with bidentate binding to Zn 2+ shows a distance of 2.7 Å. This is too far to create a H-bond and can only be considered as a dipole-dipole interaction.
The enterococcal α-CA has a deep narrow cavity (Figure 12), as is common for the α-class of this enzyme (3D representation of enterococcal carbonic anhydrase created by homology modeling is presented in Supplementary Materials Figure S4). Still, it provides enough space to create a bidentate metal-coordination bond with Zn 2+ using one of the sulfonamide oxygens, in contrast with the human CAs. The only top pose where the sulfonamide oxygen does not participate in coordination with Zn 2+ is at compound 21; however, the scoring algorithm considers the strength of the interaction only with the coulombic term because parametrization is missing for any covalent bonds. There is a high probability that the pose of 21 having a bidentate interaction with Zn 2+ is the correct one, despite the lower score. A quantum-mechanic computation method is needed to find out.  The second sulfonamide oxygen serves as an acceptor for a very weak H-bond to the backbone nitrogen of Thr-181 with a -H . . . O-distance of about 2.4 Å and, in the case of 9 R-enantiomer, also to its hydroxylic group. In the case of the bidentately-binding compound 21, the H-bond is stronger, with a distance of 1.9 Å.
All compounds have the benzenesulfonamide moiety binding to Zn 2+ in the same position except compound 21 ( Figure 11).

Retrieval of Enterococcal CA Protein Sequence
There were no resolved 3D structures available for the α-CA protein of E. faecalis in PDB yet. In the literature, it was reported that, for homology modeling, the sequence identity between target and template proteins should be 25% or more [52]. The crystal structure of CA from Thermovibrio ammonificans (4C3T) was selected with a 31% similarity sequence for the target sequence and 6% for gaps. The sequence alignment is depicted in Figure 9. In the first row is the sequence of template protein; the sequence of the target protein is in the last row.

3D Homology Modeling
The stereochemical quality of the 3D model was validated by the Ramachandran plot. Figure 10 showed that around 97.8% of the residues were present in the allowed regions (88.4% in the favored region and 9.4% residues in the allowed regions) and that only 2.2% of residues were present in the outlier region, indicating that the quality of the model was good. The plot did not change significantly after MD (Molecular dynamic) simulation, and MD showed the stability of the structure (Figure 16, see Section 4.6.4. in Materials and Figure 9. Sequence alignment. In the first row is the sequence of template protein; the sequence of the target protein is in the last row. The stereochemical quality of the 3D model was validated by the Ramachandran plot. Figure 10 showed that around 97.8% of the residues were present in the allowed regions (88.4% in the favored region and 9.4% residues in the allowed regions) and that only 2.2% of residues were present in the outlier region, indicating that the quality of the model was good. The plot did not change significantly after MD (Molecular dynamic) simulation, and MD showed the stability of the structure (Figure 16, see Section 4.6.4. in Materials and Methods). From our observation, we conclude that an increase in the inhibition activity of 32 can be achieved by substitution of the stilbene aromatic ring near s-triazine with an acidic functional group which would create a salt bridge with the primary ammonium group of Lys-185. Figure 12. Compound 32 is docked in the active site of enterococcal α-CA homology model. Color of atoms: hydrogenwhite, carbon-gray, nitrogen-blue, oxygen-red and sulfur-yellow, Zn 2+ -gray-blue sphere. Intermolecular interactions: H-bond-yellow dashed line, π-π stacking-light blue dashed line. Color of the surface of the binding site: hydrophobic residues-green, polar residues-light blue, negatively charged residues-red, positively charged residues-dark blue. Figure 12. Compound 32 is docked in the active site of enterococcal α-CA homology model. Color of atoms: hydrogen-white, carbon-gray, nitrogen-blue, oxygen-red and sulfur-yellow, Zn 2+gray-blue sphere. Intermolecular interactions: H-bond-yellow dashed line, π-π stacking-light blue dashed line. Color of the surface of the binding site: hydrophobic residues-green, polar residues-light blue, negatively charged residues-red, positively charged residues-dark blue.

Binding Energy Calculation
The values of the binding free energies of top poses obtained from the IFD output (Table 6) are in the range from −51.59 to −75.35 kcal/mol. These values predict strong binding to the enterococcal α-CA. The correlation with MIC is poor because of the very narrow range of both computed binding energies and concentrations (taking expected error of both methods into account), and other features may have an impact, e.g., penetration through a bacterial cell wall. Figure 13. Both enantiomers of compound 9 are docked in the active site of the enterococcal α-CA homology model. The color of carbon atoms for compounds 9-S is green and is teal for compound 9-R. Color of other elements: hydrogenwhite, nitrogen-blue, oxygen-red, sulfur-yellow, Zn 2+ -gray-blue sphere. Intermolecular interactions: H-bond-yellow dashed line.

Binding Energy Calculation
The values of the binding free energies of top poses obtained from the IFD output (Table 6) are in the range from −51.59 to −75.35 kcal/mol. These values predict strong binding to the enterococcal α-CA. The correlation with MIC is poor because of the very narrow range of both computed binding energies and concentrations (taking expected error of both methods into account), and other features may have an impact, e.g., penetration through a bacterial cell wall.
The S-enantiomer of compound 9 has computed a stronger binding affinity, although the chirality does not seem to play an important role in this part of the structure, as the Renantiomer seems to belong to the group with the best compounds, according to the computed energies and scores.  The S-enantiomer of compound 9 has computed a stronger binding affinity, although the chirality does not seem to play an important role in this part of the structure, as the R-enantiomer seems to belong to the group with the best compounds, according to the computed energies and scores.

Prediction of ADMET and Fluorescence
According to the predictions acquired from QikProp (Table 7), we can assume that the studied compounds would not reach the CNS. The most active compound, 32, has good oral absorption. The lower oral bioavailability of 7 and 9 is associated with a higher hydrophilicity, making those compounds more soluble in water. Caution needs to be taken because of the high predicted affinity to the Human ether-a-go-go-related gene (HERG). QikProp identified the acceptor carbonyl as a reactive group, which can explain the high experimentally measured toxicity of all chalcones. The probable metabolism would include the oxidative hydroxylation of phenyl rings in para-and meta-positions, the oxidation of hydroxyls on aliphatic carbons to carboxylic acids in the case of primary alcohols, and the ketones in the case of secondary alcohols. Table 7. Predicted octanol/water partition coefficient (QPlogP oct/w ), water solubility (QPlogS), peroral absorption in %, affinity to Human ether-a-go-go-related gene (QPlogHERG), and brain/blood partition coefficient (QPlogBB). All except oral absorption are decadic logarithms of molar concentration. According to GLORY, additional metabolic pathways are the hydroxylation of nitrogen or the neighboring carbon of the link between the s-triazine ring and benzenesulfonamide and the cleavage of the whole substituent from the s-triazine ring ( Figure 14).

Ligand
The predictions of ChemFLuo (Table 8)  The predictions of ChemFLuo (Table 8) show that all compounds except 9 could be used for fluorescent labeling of the enzyme with a green light.

Conclusions
A series of forty-four 1,3,5-triazinyl aminobenzenesulfonamides was prepared as potential carbonic anhydrase (hCA) inhibitors. All tested compounds are weak inhibitors of

Conclusions
A series of forty-four 1,3,5-triazinyl aminobenzenesulfonamides was prepared as potential carbonic anhydrase (hCA) inhibitors. All tested compounds are weak inhibitors of physiological isoenzyme hCA I and II. All the chalcone derivatives substituted with 3-OH demonstrated inhibitory activity against hCA VII; compounds 15 and 26 showed the highest activity and selectivity. On the other hand, stilbene derivatives, e.g., molecules 31 and 32, showed to be good inhibitors of isoenzyme hCA XII. Activity against VRE isolates is associated with substitution with the 1-(4-hydroxyphenyl)amino fragment and the stilbene fragment; compounds 21 and 32 demonstrated the highest activity against all tested strains. The most active compounds were evaluated for their cytotoxicity against HCT116 p53 +/+ , and, except for derivatives 7 and 32, showed no toxic effect up to a concentration of 50 µM; i.e., compounds 7 and 32 could be further tested for their possible use as anticancer drugs. In addition, compound 32 has the potential of a multi-target compound to act against both colorectal tumor and enterococcus. The most promising anti-VRE agent 21 exhibited no cytotoxic activity and could be discussed as a promising antibacterial agent. The molecular modeling and docking of active compounds into various hCA isoenzymes, including bacterial carbonic anhydrase, specifically α-CA present in VRE, was performed and outlined in a possible mechanism of selective anti-VRE activity. Thus, it can be stated that the selected derivatives described here deserve further attention and a deeper investigation of their biological properties.

General Method for Synthesis of Chalcones
4-Aminoacetophenone (1 eq; 10 mmol) was dissolved in 20 mL of methanol. Then, 1.15 eq (11.5 mmol) of benzaldehyde, 4-hydroxybenzaldehyde, 3-hydroxybenzaldehyde, or 2-hydroxybenzaldehyde was added. Finally, a catalytic amount of H 2 SO 4 and 5 mol% of Ce(III) ions supported on the weakly acidic resin were added, and the reaction mixture was refluxed until the disappearance of starting aminoacetophenone. The reaction was monitored by TLC: eluent n-hexane:ethyl-acetate = 1:1, UV light (254 and 356 nm), and ninhydrin reagent (at 180 • C) were used for the detection of spots. After the completion of the reaction, the catalyst was filtered off. The reaction mixture was concentrated on a rotary vacuum evaporator to 1/3 of the volume. The crude product was precipitated with cold water and filtered off. The precipitate was suspended in a minimal amount of cold water and added to a dropwise saturated solution of NaHCO 3 until it reached pH 7-8. The solid was filtered again and washed with cold water until it reached a neutral pH. The crude product was purified as follows: solid was dissolved in hot ethanol, then water was added until the first turbidity. The mixture was placed into the fridge (5-7 • C) overnight, and the clear product was filtered.

General Method for Synthesis of Stilbenes
4-Aminostilbene precursors were synthesized by the Wittig-Horner reaction based on [36,37].
In a three-necked flask was mixed one eq (10 mmol) of benzyl chloride or X-(chloromethyl) phenol with 1.2 eq (12 mmol) of triethyl phosphite. The reaction mixture was stirred under the argon atmosphere and heated at 150 • C for 3 h. Then, the reaction mixture was cooled in an ice bath, and 25 mL of dry DMF was added. With continuous cooling and stirring, the 1.5 eq (15 mmol) of solid NaH was added portionwise. After the completion of the added NaH, the reaction mixture was cooled and stirred for another 30 min. Then, one eq (10 mmol) of 4-amino benzaldehyde dissolved in 10 mL of dry DMF was added dropwise. Then, the reaction mixture was stirred at room temperature for another 15 h. The reaction was monitored by TLC: eluent ethyl-acetate: hexane = 1:4; UV light (254 and 356 nm), I 2 vapors, and anisidine reagent (at 180 • C) were used for the detection of spots. The pure product was obtained by pouring the reaction mixture at 100 g of crushed ice and recrystallization from methanol.
Stilbenes synthesized by the Wittig-Horner reaction have an exclusively trans configuration. [36,37]  Synthesis of X-hydroxybenzylchlorides: Commercially unavailable X-hydroxybenzylchlorides (I, J, K, L) were prepared by modified synthesis [57]. In our modification, the synthesis was carried out without the solvent, and the DMF was used as the organic catalyst. This modification achieved significantly higher %yields. In the flask equipped with the calcium chloride drying tube was cooled the mixture of 1 eq (20 mmol) of benzylalcohol or X-(hydroxymethyl)phenol and a catalytical amount of DMF in an ice bath (0-5 • C). Then, the X-(hydroxymethyl)phenol was overlayed with 3 eq (60 mmol) of SOCl 2 . The reaction mixture was vigorously stirred for 10 min under intensive cooling. Then, the reaction mixture was stirred for 1 h at room temperature. The reaction was monitored by TLC: eluent EtOH, UV light (254 and 356 nm), and anisidine reagent (at 180 • C) were used for the detection of spots. After the completion of the reaction, the reaction mixture was dissolved in 10 mL of chloroform and was washed five times with 20 mL of distilled water. The organic layer was dried with anhydrous sodium sulfate. Chloroform was evaporated at a rotary vacuum evaporator (15 mbar, 25 • C). Preparation of 4-aminobenzaldehyde: 4-Aminobenzaldehyde was synthesized according to the [58]. Obtained 1 H-NMR spectra and 13 C-NMR spectra are in agreement with those previously reported [58] Compounds (1, 2, 5-8, and 18-20) were prepared according to the methodology published in [38,39]. All spectral data of known compounds (1, 2, 5-8, and 18-20) are in agreement with those previously reported [39].
Step 1: Starting dichlorotriazinyl benzenesulfonamide (1 mmol) was dissolved in 10 mL of DMF. One mmol of solid anhydrous potassium carbonate was added gradually, and the mixture was stirred for 10 min. Then, one mmol of the appropriate nucleophile was added portionwise. Finally, 2.5% mol of supported Cu(I) ions were added into the reaction mixture. The reaction was stirred at 35 • C until the maximum conversion of starting reactants was achieved (monitored by TLC). After the completion of a reaction, the catalyst and salt were filtered off. Crushed ice was then added into the solution, and the formed precipitate was collected by filtration. The crude product was dissolved in hot acetone and precipitated by the addition of isopropyl alcohol.
Step 2: Appropriate chalcone (1 mmol) was dissolved in 15 mL of DMF. One mmol of solid anhydrous potassium carbonate was added in small portions, and the mixture was stirred for 15 min. Then, one mmol of the appropriate nucleophile (a disubstituted derivative of cyanuric chloride) was added portionwise. Finally, 2.5% mol of supported Cu(I) ions were added into the reaction mixture. The reaction was stirred at 110 • C until the maximum conversion of starting reactants was achieved (monitored by TLC). After the completion of a reaction, the catalyst and salt were filtered off. The filtrate was concentrated to 1/5 of the original volume by a rotary vacuum evaporator. The pure product was obtained by the addition of the mixture of isopropyl alcohol: diethyl ether (1:10) and filtered off.
Step 1: Starting dichlorotriazinyl benzenesulfonamide (1 mmol) was dissolved in 10 mL of DMF. One mmol of solid anhydrous potassium carbonate was added in small portions, and the mixture was stirred for 10 min. Next, 1 mmol of the appropriate nucleophile was added portionwise. Finally, 2.5% mol of supported Cu(I) ions were added into the reaction mixture. The reaction mass was stirred at 35 • C until the maximum conversion of starting reactants was achieved (monitored by TLC). After the completion of a reaction, the catalyst and salt were filtered off. Crushed ice was added into the solution, and the formed precipitate was collected by filtration. The crude product was dissolved in hot acetone and precipitated by the addition of isopropyl alcohol.
Step 2: Appropriate stilbene (1 mmol) was dissolved in 15 mL of DMF. One mmol of solid anhydrous potassium carbonate was added in small portions, and the mixture was stirred for 15 min. Then, one mmol of the appropriate nucleophile (a disubstituted derivative of cyanuric chloride) was added portionwise. Finally, 2.5% mol of supported Cu(I) ions were added to the reaction mixture. The reaction mass was stirred at 130 • C until the maximum conversion of the starting reactants was achieved (monitored by TLC). After the completion of the reaction, the catalyst and salt were filtered off. The filtrate was concentrated to 1/10 of the original volume by a rotary vacuum evaporator, and 15 mL of isopropyl alcohol was added. The mixture was cooled at 0-5 • C overnight. The obtained pure product was filtered off. The other portion of pure product was obtained as follows: The filtrate was treated with a few drops of diethyl ether, and then the solvent was evaporated entirely by a rotary vacuum evaporator. Then, 2 mL of cold water was poured into the mixture, and the crystals were formed. The mixture was cooled to 0-5 • C (in the fridge) for 72 h and was then filtered.
All spectral data of known compounds (17,35,37,39,43) are in agreement with those previously reported [40] the minimum inhibitory concentration was evaluated. A total of 20 µL of Alamar Blue solution was added to each well, and the plate was incubated 1 h at 37 • C. The minimum inhibitory concentration was evaluated as the lowest concentration of the tested compound, which fully inhibited the color change of Alamar Blue from blue to pink. Vancomycin and ampicillin were used as reference drugs. The experiment was repeated at least three times. The human colorectal tumor cell line (HCT116 p53 +/+ ) was given by Dr. Vogelstein [67]. Cells were grown in a tissue culture flask 25 cm 2 (TPP) in Dulbecco's Modified Eagle Medium (DMEM) with high glucose 4.5 g/L, L-glutamine, and natrium pyruvate (Biosera). The medium was supplemented with 10 % fetal bovine serum (Biosera, Nuaille, France) and 100 µg/mL of penicillin/streptomycin (Biosera, Nuaille, France).

MTT Assay
Cells were harvested using trypsin (Biosera, Nuaille, France) and were then seeded into the 10 cm tissue culture dish (TPP). The cells were harvested after 24 h during the exponential growth phase and were seeded into a tissue culture test plate with 96 wells (TPP) at a concentration of 7.5 × 10 3 cells per well and incubated for 24 h. After this period, the growth medium was exchanged for a medium containing compounds in a concentration range from 0.1 to 50 µM. Stock solutions of the investigated compounds were prepared in DMSO (Sigma-Aldrich, Darmstadt, Germany). After 48 h of incubation with the investigated compounds, the medium was replaced with 100 µL of DMEM and 20 µL of MTT solution in each well. This solution was prepared by dissolving 2.5 mg of the Thiazolyl Blue Tetrazolium Bromide (Sigma-Aldrich, Darmstadt, Germany) per 1 mL 1 × PBS. After 2 h of incubation, the medium with MTT solution was replaced with isopropylalcohol (150 µL to each well) to dissolve the newly created formazan crystals. After 10 min, absorbance was measured using a Synergy H1 Hybride Multi-mode Microplate Reader (Bio Tek; Agilent Technologies, Santa Clara, CA, USA) at 595 nm. The inhibitory concentration (IC 50 ) was defined as the concentration of the investigated compounds that was necessary to reduce the metabolic activity of cells to 50 % of the untreated control cells, and it was expressed as means ± standard deviation (SD) in software GraphPadPrism 5 (GraphPad Software, San Diego, CA, USA). Each individual compound was tested in triplicate and repeated three times. 4.6. Molecular Modeling 4.6.1. Molecular Docking into hCA IX The crystal structure (PDB: 3IAI [68]) of hCA IX was prepared via protein preparation wizard [69] in Maestro 12.7 (Schrödinger, Inc., Mannheim, Germany). Bond orders were assigned. All missing hydrogen atoms were added. As the hCA IX is a homotetramer, the B, C, and D chains were removed. The structure was optimized for pH 7.0 using PROPKA. All water molecules were removed. Finally, the protein was minimized using the OPLS3e force field [70], and a grid for docking was generated.
Using the CombiGlide module of Schrödinger Suite (Schrödinger, Inc., Mannheim, Germany), a small virtual combinatorial library of 76 compounds was prepared (Supplementary Materials). Ligands were prepared using the LigPrep module in Maestro. In order to obtain correct molecular geometries and protonation states at pH 7.0 ± 2.0, Epik module and OPLS3e force field [70] were used. Metal-binding states were added.
Docking was performed in the program Schrodinger Glide [71] with XP precision for all molecules. Penalization for a low probable ionization state was used. As it is well known that sulfonamide bound to CAs create a metal-coordination bond between sulfonamide nitrogen and zinc dication, and that this strong type of interaction is not in the parametrization of the scoring function, the constraint to preserve the metal-coordination bond between zinc dication and deprotonated sulfonamide group was specified. A sam-pling of the nitrogen inversions and ring conformation was allowed. Relative binding affinities with optimization of poses and residues of amino acids within 5 Å for all docked poses were predicted for the most interesting ligands by Prime/MM-GBSA method [72,73]. 4.6.2. Retrieval of Enterococcal CA Protein Sequence E. faecalis contains an α-CA and a γ-CA. The γ-CAs have a much smaller active site cavity and are much less efficient catalyst [6], making the α-CA a probable target of studied sulfonamides. The spatial structure of enterococcal α-CA was not resolved by now, therefore homology modeling of the enzyme was needed. The sequence of the α-CA from E. faecalis ATCC 29212, which was used in the screening, was retrieved from the NCBI database [74] with accession number OOC96771 and 232 amino acid length [75].

3D Homology Modeling of Enterococcal CA
The suitable template for homology modeling was identified in PDB using their advanced search to identify the highest sequence identity protein. The 3D model of the enterococcal α-CA was built using Prime in Schrodinger Suite 2018-4 [72,73] using the identified protein as a template. The target and template sequences were aligned using the Clustal W method in Prime. The overall stability of the protein was assessed using the Ramachandran plot [76].

Molecular Dynamic (MD) Simulation of Enterococcal CA
Structure 32 (with the lowest measured MIC against E. faecalis) was docked into the active site of the modeled 3D structure of the enterococcal α-CA using Glide with XP protocol, and with the best pose found was further optimized using MD simulations. Molecular dynamics and molecular mechanics studies were designed and performed utilizing Gromacs simulation suite, version 2018.5. Initial structural inspections and visualizations were administered to Pymol 2.0.7 and Visual Molecular Dynamics (VMD). Molecular dynamics simulation of enterococcal protein was carried out with the inclusion of an explicit TIP3P water model in order to enable relaxation of structure required for the further production stage of analysis. We also performed MD equilibration and production runs for enzyme structure in a vacuum (without any solvent). Atomistic simulations and minimizations were carried out under the potential field of a CHARMM36 all-atom forcefield, which is suitable for modeling interactions regarding metalloproteins [78].
Prior to MD simulation, geometry optimization was carried out to avoid steric clashes and to lead the system to local minima. The structure of the protein was confined to periodic boundary conditions of the dodecahedron box with 10,636 water molecules and 8 chlorine atoms to balance the net charge of the system. The distance of the receptor of 2 nm from each box boundary is maintained to prevent protein self-interaction. Geometry optimization protocol consisted of the steepest descent algorithm with at least 1000 kcal/mol/nm maximum force necessary to stop minimization and optimization step of 0.01. The maximum number of minimization steps was set to 5000. The optimization process is depicted in Figure 15.
Following the energy minimization, we approached to MD simulations with initial 500 ps equilibration in NVT ensemble, where N-number of particles, V-volume, and T-temperature were conserved. The production stage of 10 ns simulation was subjected to NPT ensemble (N-number of particles, P-pressure, and T-temperature where conserved). According to the analysis of the root mean square deviation (RMSD) of the protein structure, we decided to stop the production phase after 10 ns. As one can see in Figure 16, RMSD oscillates around the mean value.
1 Figure 15. Energy minimization progress. The graph illustrates the descent of potential energy of receptor + solvent system as steepest descent algorithm is applied with the aforementioned conditions.  Figure 15. Energy minimization progress. The graph illustrates the descent of potential energy receptor + solvent system as steepest descent algorithm is applied with the aforementioned con tions. Figure 16. RMSD of whole protein structure after 10 ns production run. The whole 10 ns portion of the run was used for analysis. Only marginal variation of RMSD during 10 ns around the mean value of~0.035 nm can be observed, suggesting the stability of the receptor structure after NVT equilibration run and within NPT production MD run.
A portion of the final 1 ns trajectory was further utilized for analysis. The integration time step was set to 2 fs, and every 1 ps frame of calculated trajectory was stored. Leapfrog integrator was employed with NVT and also an NPT production run ensemble. The periodic boundary conditions (PBC) were applied isotropically in all directions. The receptor and solvent molecules were thermostated at 298 K by the Parrinello-Rahman velocity rescale algorithm [79]. Isotropic pressure was applied by the Berendsen algorithm with an equilibrium pressure of 1 bar with a time constant of 3.0 ps and compressibility of 4.5 × 10 −5 bar −1 [80]. The short-range cut-off for Lennard-Jones and electrostatic interactions were regulated to 10 Å. The long-range interactions with the reciprocal-space interactions evaluated on a 0.16 nm spacing grid and with cubic interpolation of fourthorder were treated by particle mesh Ewald (PME) method. LINCS algorithm was employed to constraint all bond lengths [81]. The 3D representation of prepared protein structure is in Figure 4 The five compounds most active against E. faecalis (7, 9, 21, 25, 32) were used in the molecular docking exploiting Induced Fit Docking (IFD) protocol of Schrödinger Suite [82]. The IFD protocol involves the use of both the Glide docking program [71] and the Prime [72,73], a module for protein structure modeling. We chose the IFD procedure to implement flexibility of protein, as we did not have enough information about the conformation of the binding site amino acid residues when inhibitors are bound for this protein. No sidechains of the protein homology model were trimmed, just the van der Waals radii of ligand and protein atoms were scaled to half of their normal size during initial docking. The ligand molecules were prepared for IFD using LigPrep to generate all possible tautomers, enantiomers, and ionization states at pH 7.0 ± 2.0 using Epik [83,84]. All molecules are deprotonated on the sulfonamide group as it is well known that primary sulfonamide creates a complex bond with zinc dication (Zn 2+ ) through the deprotonated amine of the sulfonamide group [85]. Afterward, were all generated molecules minimized using the OPLS 3e forcefield [70]. The best pose is chosen based on the IFD score calculated according to the following formula: IFDScore = 1.0 * Prime_Energy + 9.057 * GlideScore + 1.428 * Glide_Ecoul, where Prime_Energy is the energy of protein calculated with Prime, GlideScore is the score calculated with docking module Glide, and Glide_Ecoul is the Coulomb term of the GlideScore. 4.6.6. Binding Energy Calculation with Enterococcal CA The binding free energies of top poses obtained from IFD output were carried out by using Prime-MM/GBSA (molecular mechanics generalized Born surface area). Prime MM/GBSA includes the OPLS 3e force field [70], VSGB solvent model [77], and rotamer searching algorithms. The MM/GBSA calculations are used to estimate the relative binding affinity of ligands to the binding site of the protein (reported in kcal/mol). As the MM/GBSA binding energies are approximate free energies of binding, a more negative value indicates stronger binding.

Prediction of ADMET and Fluorescence
Except for finding important structural features for binding with the target protein, it was in our interest to look for the ADMET properties of the most promising compounds. Schrodinger QikProp was used to calculate the ADMET profile for the five s-triazine analogues, and GLORY [86,87] was used to additional predictions of metabolism.
Stilbenes and especially chalcones are known to have fluorescent properties. Prediction tool ChemFLuo [88] was used to predict the probability of the compounds to possess green or blue fluorescence.